An unlabeled probe based real time PCR and modified semi nested PCR as molecular tools for analysis of chloroquine resistant in Plasmodium vivax isolates from Afghanistan

Abstract Background: Chloroquine (CQ) resistance Plasmodium vivax isolates have been reported from many endemic regions in the world. P. vivax has been reported to be about 95% of the whole malaria in Afghanistan and CQ is prescribing in the first-line treatment of vivax malaria. The pvmdr-1 and pvcrt-o (K10 insertion) genes are the possible markers of CQ-resistance P. vivax isolates. There have been no studies done on the prevalence of molecular markers of CQ-resistance P. vivax in Afghanistan. In the present work, we aimed to evaluate the prevalence of mutations in the pvmdr-1 and K10 insertion in the pvcrt-o genes of P. vivax.Methods: P. vivax isolates were collected from Laghman, Baghlan and Khost provinces. For investigation of polymorphisms of desired regions in pvmdr-1 and pvcrt-o genes, sequencing was applied on the PCR products. We developed a new asymmetric qPCR and melting analysis assay based on unlabeled probe for scanning of K10 insertion in pvcrt-o gene.Results: The analysis of sequencing data of the pvmdr-1 gene showed wild type Y976 and K997 and mutant M958 and L1076 in 33 isolates from three provinces and submitted in GenBank (accession number MK419882-MK419914)Of 36 samples that evaluated for K10 insertion in pvcrt-o, 2/18(11%), 0/10(0%) and 0/8(0%) isolates from Laghman, Baghlan and Khost province possessed K10 insertion, respectively, that confirmed by either sequencing and unlabeled probes and submitted in GenBank (accession number MK292011-MK292046).Conclusion: The existence of 2 samples with K10 insertion and 33 samples with pvmdr1 polymorphism indicating on CQ resistance P. vivax populations in Afghanistan. This can lead to spreading of resistant strains in the society. Furthermore, unlabeled probes are simple and inexpensive alternative tools for screening of P. vivax mutations.

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An unlabeled probe based real time PCR and modified semi nested PCR as molecular tools for analysis of chloroquine resistant in Plasmodium vivax isolates from Afghanistan

10.21203/rs.2.24527/v2 ◽

2020 ◽

Author(s):

Sayed Hussain Mosawi ◽

Abdolhossein Dalimi ◽

Najibullah Safi ◽

Reza Fotouhi-Ardakani ◽

Fatemeh Ghaffarifar ◽

...

Keyword(s):

Plasmodium Vivax ◽

Molecular Tools ◽

Wild Type ◽

Accession Number ◽

Sequencing Data ◽

First Line ◽

Unlabeled Probe ◽

Two Samples ◽

Pcr Products ◽

Melting Analysis

Abstract Background: Plasmodium vivax isolate resistance to Chloroquine (CQ) has been reported from many endemic regions in the world. P. vivax is responsible for 95% of malaria cases in Afghanistan and CQ is the first-line treatment given for treatment of vivax malaria. The pvmdr-1 and pvcrt-o (K10 insertion) genes are the possible markers for CQ-resistance P. vivax isolates. There have been no studies done on the presence or absence of molecular markers for CQ-resistance P. vivax in Afghanistan. In the present work, we aimed to evaluate the frequency of mutations in the pvmdr-1 and K10 insertion in the pvcrt-o genes of P. vivax.Methods: P. vivax isolates were collected from Laghman, Baghlan and Khost provinces. For investigation of polymorphisms of desired regions in pvmdr-1 and pvcrt-o genes, sequencing was applied on the PCR products. We developed a new asymmetric qPCR and melting analysis assay based on unlabeled probe for scanning of K10 insertion in pvcrt-o gene. Results: The analysis of sequencing data of the pvmdr-1 gene showed wild type Y976 and K997 and mutant M958 and L1076 in 33 isolates from three provinces and submitted in GenBank (accession number MK419882-MK419914)Of 36 samples that evaluated for K10 insertion in pvcrt-o, 2/18(11%), 0/10(0%) and 0/8(0%) isolates from Laghman, Baghlan and Khost province possessed K10 insertion, respectively, that confirmed by either sequencing and unlabeled probes and submitted in GenBank (accession number MK292011-MK292046).Conclusion: Two samples with K10 insertion and 33 samples with pvmdr1 polymorphism, indicating on the possibility of CQ resistance P. vivax populations in Afghanistan. Furthermore, unlabeled probes are simple and inexpensive alternative tools for screening of P. vivax mutations.

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An unlabeled probe based real time PCR and modified semi nested PCR as molecular tools for analysis of chloroquine resistant in Plasmodium vivax isolates from Afghanistan

10.21203/rs.2.24527/v3 ◽

2020 ◽

Author(s):

Sayed Hussain Mosawi ◽

Abdolhossein Dalimi ◽

Najibullah Safi ◽

Reza Fotouhi-Ardakani ◽

Fatemeh Ghaffarifar ◽

...

Keyword(s):

Plasmodium Vivax ◽

Molecular Tools ◽

Wild Type ◽

Accession Number ◽

Sequencing Data ◽

First Line ◽

Unlabeled Probe ◽

Two Samples ◽

Pcr Products ◽

Melting Analysis

Abstract Background: Plasmodium vivax isolate resistance to Chloroquine (CQ) has been reported from many endemic regions in the world. P. vivax is responsible for 95% of malaria cases in Afghanistan and CQ is the first-line treatment given for treatment of vivax malaria. The pvmdr-1 and pvcrt-o (K10 insertion) genes are the possible markers for CQ-resistance P. vivax isolates. There have been no studies done on the presence or absence of molecular markers for CQ-resistance P. vivax in Afghanistan. The present work aimed to evaluate the frequency of mutations in the pvmdr-1 and K10 insertion in the pvcrt-o genes of P. vivax.Methods: P. vivax isolates were collected from Laghman, Baghlan and Khost provinces. For investigation of polymorphisms of desired regions in pvmdr-1 and pvcrt-o genes, sequencing was applied on the PCR products. A new asymmetric qPCR and melting analysis assay based on unlabeled probe developed for scanning of K10 insertion in pvcrt-o gene. Results: The analysis of sequencing data of the pvmdr-1 gene showed wild type Y976 and K997 and mutant M958 and L1076 in 33 isolates from three provinces and submitted in GenBank (accession number MK419882-MK419914)Of 36 samples that evaluated for K10 insertion in pvcrt-o, 2/18(11%), 0/10(0%) and 0/8(0%) isolates from Laghman, Baghlan and Khost province possessed K10 insertion, respectively, that confirmed by either sequencing and unlabeled probes and submitted in GenBank (accession number MK292011-MK292046).Conclusion: Two samples with K10 insertion and 33 samples with pvmdr1 polymorphism, indicating on the possibility of CQ resistance P. vivax populations in Afghanistan. Furthermore, unlabeled probes are simple and inexpensive alternative tools for screening of P. vivax mutations.

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An unlabelled probe-based real time PCR and modified semi-nested PCR as molecular tools for analysis of chloroquine resistant Plasmodium vivax isolates from Afghanistan

10.21203/rs.2.24527/v4 ◽

2020 ◽

Author(s):

Sayed Hussain Mosawi ◽

Abdolhossein Dalimi ◽

Najibullah Safi ◽

Reza Fotouhi-Ardakani ◽

Fatemeh Ghaffarifar ◽

...

Keyword(s):

Plasmodium Vivax ◽

Molecular Tools ◽

Wild Type ◽

Sequencing Data ◽

First Line ◽

Two Samples ◽

The World ◽

Pcr Products ◽

Melting Analysis ◽

First Line Treatment

Abstract Background Plasmodium vivax resistance to chloroquine (CQ) has been reported from many endemic regions in the world. Plasmodium vivax is responsible for 95% of malaria cases in Afghanistan and CQ is the first-line treatment given for treatment of vivax malaria. The pvmdr-1 and pvcrt-o (K10 insertion) genes are possible markers for CQ-resistance in P. vivax isolates. There have been no studies done on the presence or absence of molecular markers for CQ-resistance P. vivax in Afghanistan. The present work aimed to evaluate the frequency of mutations in the pvmdr-1 and K10 insertion in the pvcrt-o genes of P. vivax.Methods Plasmodium vivax isolates were collected from Laghman, Baghlan and Khost provinces. For investigation of polymorphisms of desired regions in pvmdr-1 and pvcrt-o genes, sequencing was applied on the PCR products. A new asymmetric qPCR and melting analysis assay based on unlabelled probe developed for scanning of K10 insertion in pvcrt-o gene. Results The analysis of sequencing data of the pvmdr-1 gene showed wild type Y976 and K997 and mutant M958 and L1076 in 33 isolates from three provinces. Of the 36 samples evaluated for K10 insertion in pvcrt-o, 2/18(11%), 0/10(0%) and 0/8(0%) isolates from Laghman, Baghlan and Khost province, respectively, possessed K10 insertion, confirmed by either sequencing and unlabelled probes.Conclusion Two samples with K10 insertion and 33 samples with pvmdr1 polymorphism, indicating on the possibility of CQ resistance in P. vivax populations in Afghanistan. Furthermore, unlabelled probes are simple and inexpensive alternative tools for screening of P. vivax mutations.

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An unlabelled probe-based real time PCR and modified semi-nested PCR as molecular tools for analysis of chloroquine resistant Plasmodium vivax isolates from Afghanistan

10.21203/rs.2.24527/v5 ◽

2020 ◽

Author(s):

Sayed Hussain Mosawi ◽

Abdolhossein Dalimi ◽

Najibullah Safi ◽

Reza Fotouhi-Ardakani ◽

Fatemeh Ghaffarifar ◽

...

Keyword(s):

Plasmodium Vivax ◽

Molecular Tools ◽

Wild Type ◽

Sequencing Data ◽

First Line ◽

Two Samples ◽

The World ◽

Pcr Products ◽

Melting Analysis ◽

First Line Treatment

Abstract Background Plasmodium vivax resistance to chloroquine (CQ) has been reported from many endemic regions in the world. Plasmodium vivax is responsible for 95% of malaria cases in Afghanistan and CQ is the first-line treatment given for treatment of vivax malaria. The pvmdr-1 and pvcrt-o (K10 insertion) genes are possible markers for CQ-resistance in P. vivax isolates. There have been no studies done on the presence or absence of molecular markers for CQ-resistance P. vivax in Afghanistan. The present work aimed to evaluate the frequency of mutations in the pvmdr-1 and K10 insertion in the pvcrt-o genes of P. vivax. Methods Plasmodium vivax isolates were collected from Laghman, Baghlan and Khost provinces. For investigation of polymorphisms of desired regions in pvmdr-1 and pvcrt-o genes, sequencing was applied on the PCR products. A new asymmetric qPCR and melting analysis assay based on unlabelled probe developed for scanning of K10 insertion in pvcrt-o gene. Results The analysis of sequencing data of the pvmdr-1 gene showed wild type Y976 and K997 and mutant M958 and L1076 in 33 isolates from three provinces. Of the 36 samples evaluated for K10 insertion in pvcrt-o , 2/18(11%), 0/10(0%) and 0/8(0%) isolates from Laghman, Baghlan and Khost province, respectively, possessed K10 insertion, confirmed by either sequencing and unlabelled probes. Conclusion Two samples with K10 insertion and 33 samples with pvmdr1 polymorphism, indicating on the possibility of CQ resistance in P. vivax populations in Afghanistan. Furthermore, unlabelled probes are simple and inexpensive alternative tools for screening of P. vivax mutations.

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Sensitivity and Specificity of Single-Nucleotide Polymorphism Scanning by High-Resolution Melting Analysis

Clinical Chemistry ◽

10.1373/clinchem.2003.029751 ◽

2004 ◽

Vol 50 (10) ◽

pp. 1748-1754 ◽

Cited By ~ 368

Author(s):

Gudrun H Reed ◽

Carl T Wittwer

Keyword(s):

Single Nucleotide Polymorphism ◽

High Resolution ◽

Sensitivity And Specificity ◽

High Resolution Melting ◽

Wild Type ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Product Size ◽

Pcr Products ◽

Melting Analysis

Abstract Background: Screening for heterozygous sequence changes in PCR products, also known as “mutation scanning”, is an important tool for genetic research and clinical applications. Conventional methods require a separation step. Methods: We evaluated the sensitivity and specificity of homogeneous scanning, using a saturating DNA dye and high-resolution melting. Heterozygous single-nucleotide polymorphism (SNP) detection was studied in three different sequence backgrounds of 40%, 50%, and 60% GC content. PCR products of 50–1000 bp were generated in the presence of LCGreen™ I. After fluorescence normalization and temperature overlay, melting curve shape was used to judge the presence or absence of heterozygotes among 1632 cases. Results: For PCR products of 300 bp or less, all 280 heterozygous and 296 wild-type cases were correctly called without error. In 672 cases between 400 and 1000 bp with the mutation centered, the sensitivity and specificity were 96.1% and 99.4%, respectively. When the sequence background and product size with the greatest error rate were used, the sensitivity of off-center SNPs (384 cases) was 95.6% with a specificity of 99.4%. Most false negatives occurred with SNPs that were compared with an A or T wild type sequence. Conclusions: High-resolution melting analysis with the dye LCGreen I identifies heterozygous single-base changes in PCR products with a sensitivity and specificity comparable or superior to nonhomogeneous techniques. The error rate of scanning depends on the PCR product size and the type of base change, but not on the position of the SNP. The technique requires only PCR reagents, the dye LCGreen I, and 1–2 min of closed-tube, post-PCR analysis.

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Reevaluation of the Critical Concentration for Drug Susceptibility Testing of Mycobacterium tuberculosis against Pyrazinamide Using Wild-Type MIC Distributions andpncAGene Sequencing

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05894-11 ◽

2011 ◽

Vol 56 (3) ◽

pp. 1253-1257 ◽

Cited By ~ 35

Author(s):

J. Werngren ◽

E. Sturegård ◽

P. Juréen ◽

K. Ängeby ◽

S. Hoffner ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Critical Concentration ◽

Gene Mutations ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Clinical Isolates ◽

Wild Type ◽

First Line ◽

Content Type ◽

Resistant Strains

ABSTRACTPyrazinamide (PZA) is a potent first-line agent for the treatment of tuberculosis (TB) with activity also against a significant part of drug-resistantMycobacterium tuberculosisstrains. Since PZA is active only at acid pH, testing for susceptibility to PZA is difficult and insufficiently reproducible. The recommended critical concentration for PZA susceptibility (MIC, 100 mg/liter) used in the Bactec systems (460 and MGIT 960) has not been critically evaluated against wild-type MIC distributions in clinical isolates ofMycobacterium tuberculosis. Using the Bactec MGIT 960 system, we determined the PZA MICs for 46 clinicalM. tuberculosisisolates and compared the results topncAsequencing and previously obtained Bactec 460 data. For consecutive clinical isolates (n= 15), the epidemiological wild-type cutoff (ECOFF) for PZA was 64 mg/liter (MIC distribution range, ≤8 to 64 mg/liter), and nopncAgene mutations were detected. In strains resistant in both Bactec systems (n= 18), the PZA MICs ranged from 256 to ≥1,024 mg/liter. The discordances betweenpncAsequencing, susceptibility results in Bactec 460, and MIC determinations in Bactec MGIT 960 were mainly observed in strains with MICs close to or at the ECOFF. We conclude that in general, wild-type and resistant strains were clearly separated and correlated topncAmutations, although some isolates with MICs close to the ECOFF cause reproducibility problems within and between methods. To solve this issue, we suggest that isolates with MICs of ≤64 mg/liter be classified susceptible, that an intermediary category be introduced at 128 mg/liter, and that strains with MICs of >128 mg/liter be classified resistant.

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Saturation of Pfdhfr and Pfdhps haplotypes in general population increasingly threatens the benefits of using sulfadoxine‑pyrimethamine for intermittent preventive treatment in pregnant women in Tanzania

10.21203/rs.2.20112/v1 ◽

2020 ◽

Author(s):

George Bwire ◽

Wigilya P. Mikomangwa ◽

Manase Kilonzi

Keyword(s):

Reference Genome ◽

Intermittent Preventive Treatment ◽

Preventive Treatment ◽

Dried Blood Spots ◽

Wild Type ◽

First Line ◽

High Prevalence ◽

Finger Prick ◽

Pcr Products ◽

First Line Treatment

Abstract Background : High levels of Plasmodium falciparum resistance prompted withdrawal of sulfadoxine-pyrimethamine (SP) as the first-line treatment for uncomplicated malaria in Tanzania. However, SP was limited for intermittent preventive treatment during pregnancy (IPTp) especially where there is moderate to high malaria transmission. This study reports the patterns of P. falciparum dihydrofolate reductase ( Pfdhfr ) and dihydropteroate synthetase ( Pfdhps ) mutations. Methods: Parasite genomic DNA was extracted from dried blood spots prepared by finger prick. Batched samples (384) were sequenced in a single MiSeq lane combining all PCR products. Samples were de-plexed using the multiplexing adapters and individual CRAM files were aligned to a modified amplicon reference genome. Genotyping of Pfdhfr and Pfdhps mutations were done using bcftools as well as custom scripts to filter and translate genotypes into drug resistance haplotypes. Results: The Pfdhfr was analyzed from 445 samples, the wild type (WT) Pfdhfr haplotype NCSI was detected in only six (1.3%) samples. Triple Pfdhfr IRN I haplotype was dominant, contributing to 84% (n=374) of haplotypes. The total of 446 samples were studied for Pfdhps . WT for Pfdhps was found in 6.7% (n=30) of all samples detected. Double Pfdhps haplotype (S GE AA) accounted for 83% of mutations of the Pfdhps gene. The overall prevalence of K540E was 90.4% (n=396) while A581G was 1.1% (n=5). Additionally, 91.4% (n=447) genotypes where detected from 489 sequenced samples. Of 447 genotypes detected only 0.9% (n=4) of samples were WT (SAKAA-NCSI). Quintuple mutation, S GE AA- IRN I accounted 71.4% of concomitant Pfdhfr/Pfdhps mutations where 0.2% (n=1) had septuple mutation, AG K GS - IRN I. Conclusions : Despite the high prevalence of mutations in Pfdhfr and Pfdhps gene but the current mutations at Pfdhfr K540E and Pfdhps A581G are within the recommended WHO range, stopping IPTp-SP is recommended in areas where the Pfdhfr K540E prevalence is >95 % and Pfdhps A581G is >10 % as SP is likely to be ineffective). Nevertheless, saturation in Pfdhfr and Pfdhps haplotypes is alarming, therefore screening for alternative antimalarial drug for IPTp-SP is recommended.

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